Regulation of eating behavior

ABSTRACT

MCH and anlogs thereof in the regulation of eating and weight.

BACKGROUND OF THE INVENTION

[0001] The invention relates to MCH, MCH agonists and antagonists, andtheir use to regulate eating behavior.

[0002] Although our understanding of rodent obesity has increasedsignificantly through molecular analysis of obese mouse models, e.g.,the ob/ob mouse, agouti mouse and brown adipose tissue deficient mousemodels, the mechanism by which various molecular defects lead to alteredfeeding behavior remain largely unknown. In general, the molecularcauses of hyperphagia and obesity in both man and animals are poorlyunderstood (Bray (1989) Am. J. Clin. Nutr. 891-902). Recently twoobesity related genes have been identified using positional cloning(Zhang et al. (1994) Nature 372:425-432).

[0003] Neurotransmitters and neuropeptides are known to affect feedingbehavior. The neurotransmitters, serotonin and norepinephrine (NE) areinvolved in regulation of appetite, with serotonin agonists inhibitingappetite while NE agonists induce eating. Abnormal responses to theneurotransmitter GABA have also been reported (Tsuji et al. (1991) BrainResearch 48-54. In addition, a number of neuropeptides have beenimplicated in regulating food intake. Neuropeptide Y (NPY) mimics theaction of NE in the central nervous system. Injection of NPY inducesfeeding behavior in sated rats (Stanley et al. (1989) Physiol and Behav.46:173-177) and levels of NPY and preproNPY mRNA may be altered in obeserodents, however, the results reported from different investigators hasnot been consistent. Increased levels of NPY are seen in thehypothalamus of Wistar rats with diet-induced obesity, although mRNAlevels are unchanged (Wilding et al. (1992) J. Endocrinol. 132-299-404).In Zucker rats, NPY increased levels of hypothalamic NPY and preproNPYmRNA have been reported (Sanacor et al. (1990) Endocrinology127:730-736; Pesonen et al. (1992) 255-260; and Williams et al. (1991)Clincial Science 80:419-426). In the same model, other investigatorshave found NPY levels unchanged, in the basal state, but increasing withfood restrictions (Williams et al. (1991) Clincial Science 80:419-426).In ob/ob mice, hypothalamic NPY concentrations are unchanged, however,food restriction activates NPY gene expression (Wilding et al. (1993)Endocrinology 132:1939-1943). A number of other peptides includinggalanin, beta-endorphin, dynorphin, act to stimulate food intake insatiated rats when injected into the paraventricular nucleus orventromedian nucleus. Factors derived from the gastrointestinal tractmay also be important in appetite regulation. For example,cholecystokinin and bombesin inhibit food intake when injected into rats(Baile et al. (1986) Physiol Reviews 66:172-234 and Morley (1987)Endocrinol Rev. 8:256-87).

[0004] The presence of melanocyte concentrating hormone (MCH), a cyclicpeptide, in fish hypothalamus was described over a decade ago. The roleof MCH, in teleost fish appears to be regulation of color change; MCHinduces melanophore aggregation and MSH induces melanophore dispersion(Nahon et al. (1993) Ann. NY Acad. Sci. 680:111-129). In 1985,immunoreactive MCH was also found in rodent brain (Skofitsch et al.(1985) Brain Res. Bull. 15:635-639), and it's subcellular localizationin the rats and man was described a few years later (Naito et al. (1988)Cell Tissue Res. 253:291-295 and Bresson et al. (1987) CR Soc Biol.181:376-382). In mammals, MCH gene expression is localized to theventral aspect of the Zona Incerta and the lateral hypothalamus (Bretonet al. (1993) Molecular and Cellular Neurosciences 4:271-284). The geneencodes an MCH peptide, as well as, a 13 amino acid peptide which isprocessed and released by hypothalamic cells in culture (Parkes et al(1992) Endocrinology 131:1826-1831). MCH perikarya project throughoutthe mammalian brain, and it is likely that MCH is involved inintegrative processes which accompany complex behaviors (Skofitch et al.(1985) Brain Res. Bull. 15:635-639) (Zhang et al. (1994) Nature372:425-432).

SUMMARY OF THE INVENTION

[0005] In general, the invention features, a method of promoting eating,appetite, or the gain or maintenance of weight, in a subject including:administering an effective amount of MCH, or an agonist or fragmentthereof, to the subject.

[0006] In preferred embodiments: the subject is a mammal, e.g., a human;the subject is underweight or exhibits less than normal eating behavior;the subject suffers from an immune system disorder, e.g. AIDS, or is HIVpositive; the subject suffers from anorexia nervosa, or renal disease,e.g., chronic renal disease or renal disease requiring dialysis; thesubject is, has been, or will be, administered a treatment which resultsin decreased appetite or eating behavior or in a loss of weight, e.g.,chemotherapy, radiation therapy, or dialysis.

[0007] In preferred embodiments, the method further includes diagnosingthe subject as being at risk for: a disorder or unwanted conditionrelated to MCH metabolism; an eating, appetite, or weight-relateddisorder; less than normal eating behavior; wasting; or beingunderweight.

[0008] In preferred embodiments, the method further includes repeatingthe administration of MCH, or an agonist, or a fragment thereof.

[0009] In preferred embodiments, the method further includesadministering a treatment which results in decreased eating behavior orin a loss of weight, e.g., chemotherapy, radiation therapy, or dialysis.The treatment can be administered before, after, or during MCH or MCHagonist or fragment administration.

[0010] In preferred embodiments: the subject is a non-human animal,e.g., a non-human mammal, e.g., a non-human primate, a dog, or a rodent,e.g., a rat or a mouse; the subject is other than a fish. The subjectcan be wild type with respect to genes which condition weight or eatingbehavior, or the subject can carry one or more genetic lesions whichaffect weight or eating behavior. For example, the subject can carry amutation in the ob gene, the MCH gene, or the ob receptor gene. Thesubject can be a transgenic animal, e.g., a transgenic whichmisexpresses the ob transgene, the MCH transgene, or the ob receptortransgene. The subject can also be deficient for brown fat tissue. E.g.,a brown fat tissue “knockout” mouse can be made by fusing diphtheriatoxin to a brown fat-specific promoter.

[0011] The administration of MCH or an MCH agonist or fragment can beinitiated: when the recipient begins to show signs of insufficienteating, loss of appetite, or loss of weight, e.g., as evidenced by adecline weight of more than 10, 20, or 30% in body weight or when thesubject is 10, 20, or 30% below normal body weight; when a loss inappetite is diagnosed; at the time a treatment which inhibits eating,appetite, or weight gain or maintenance, is begun or begins to exert itseffects; or generally, as is needed to maintain health or appropriateweight levels.

[0012] The period over which the agent is administered (or the periodover which clinically effective levels are maintained in the subject)can be long term, e.g., for six months or more or a year or more, orshort term, e.g., for less than a year, more preferably six months orless, more preferably one month or less, and more preferably two weeksor less.

[0013] In another aspect, the invention features, a method of inhibitingeating, inhibiting appetite, or promoting the loss of weight, in asubject including: administering an effective amount of an antagonist ofMCH to the subject.

[0014] In preferred embodiments: the subject is a mammal, e.g., a human;the subject is overweight or exhibits compulsive or other unwantedeating behavior; the subject is, has been, or will be, administered atreatment which results in increased eating behavior, e.g., steroidtherapy.

[0015] In preferred embodiments, the method further includes diagnosingthe subject as being at risk for any of: a disorder or unwantedcondition related to MCH metabolism; an eating or weight-relateddisorder; compulsive or other unwanted eating behavior, obesity; orother eating or weight related disorder.

[0016] In preferred embodiments, the method further includes repeatingthe administration of an MCH antagonist.

[0017] In preferred embodiments, the method further includesadministering a treatment which results in increased eating behavior orin a gain of weight, e.g., steroid therapy. The treatment can beadministered before, after, or during MCH antagonist.

[0018] In preferred embodiments: the subject is a non-human animal,e.g., a non-human mammal, e.g., a non-human primate, dog, or a rodent,e.g., a rat or a mouse; the subject is other than a fish. The subjectcan be wild type with respect to genes which condition weight or eatingbehavior, or the subject can carry one or more genetic lesions whichaffect weight or eating behavior. For example, the subject can carry amutation in the ob gene, the MCH gene, or the ob receptor gene. Thesubject can be a transgenic animal, e.g., a transgenic whichmisexpresses the ob transgene, the MCH transgene, or the ob receptortransgene. The subject can also be deficient for brown fat tissue. E.g.,a brown fat tissue “knockout” mouse can be made by fusing diphtheriatoxin to a brown fat-specific promoter.

[0019] In preferred embodiments, the antagonist is a peptide analog ofMCH having at least 0.50, 60, 70, 80, or 90% homology with MCH.

[0020] The administration of an MCH antagonist can be initiated: whenthe recipient begins to show signs of unwanted eating behavior or gainin weight, e.g., as evidenced by an increase of more than 10, 20, or 30%in body weight or when the subject is 10, 20, or 30% above normal bodyweight; when an increase in appetite is diagnosed; at the time atreatment which promotes eating, appetite, or weight gain ormaintenance, is begun or begins to exert its effects; or generally, asis needed to maintain health or acceptable weight levels.

[0021] The period over which the agent is administered (or the periodover which clinically effective levels are maintained in the subject)can be long term, e.g., for six months or more or a year or more, orshort term, e.g., for less than a year, more preferably six months orless, more preferably one month or less, and more preferably two weeksor less.

[0022] The inventor has discovered that MCH induces eating behavior. Theinvention includes a number of methods for evaluating treatments oragents for MCH agonist or antagonist activity. Some methods use in vitroassays, while others use cells, and yet others use animals. Methodsreferred to herein can be used individually, or in combination, toevaluate agents for MCH agonist or antagonist activity. For example,relatively rapid in vitro or cell based assays can be used as an initialscreen and an animal assay used as a secondary screen

[0023] The treatment can be any treatment which can result in thedesired effect but the administration of agents, e.g., drugs orchemicals, is preferred. Preferably, the treatment is other thansurgical intervention, e.g., the production of surgical lesions, e.g.,electrolytic lesions, e.g., the treatment is other than a ventromedialhypothalmic lesion, e.g., an electrolytically produced ventromedialhypothalmic lesion. The agent which is evaluated can be, e.g., apolysaccharide, a nucleic acid, a fat, polypeptide, or apeptide-mimetic. Amino-acid based agents can share sequence homologywith MCH or can be unrelated by sequence homology. E.g., the agent canhave 50, 60, 70, 80, 90 or 95% homology with MCH. The agent can be alinear or cyclic peptide.

[0024] Accordingly, in another aspect, the invention features a methodof evaluating a treatment, e.g., the administration of an agent, for itseffect on eating behavior, appetite, or the maintenance of weight. Themethod includes: administering the treatment to a melanocyte basedsystem, e.g., the one or more of the frog or lizard skin, fish scale, orfish skin assays described herein, and determining if there is a changein the system, and (optionally) administering the treatment to a secondtest systemand determining the effect of the treatment on a parameterrelated to eating behavior, appetite, or weight gain or loss in thesecond system. The second system can be the same or different from thefirst. In preferred embodiments the second system is a cell-based assy,e.g., an assay which uses: a fish cell; a reptilian cell; an amphibiancell; a mammalian cell; a rodent cell, e.g., a mouse or rat cell; aprimate cell; or a human cell. In preferred embodiments: the cell is aneuronal cell, e.g., a GH3 cell, a PC12 cell, or a primary hypothalmicculture cell. In other preferred embodiments the second system is ananimal based system, e.g., the treatment is administered to an animaland the effect on a parameter related to eating behavior, e.g., eatingbehavior itself, is evaluated.

[0025] In another aspect, the invention features a method of evaluatinga treatment, e.g., the administration of an agent, for its effect oneating behavior, appetite, or the maintenance of weight. The methodincludes: providing an animal, cell (an animal, plant, or bacterialcell), or cell culture preparation, having a reporter gene linked to thepromoter region of MCH; administering the treatment; and determining ifthere is an effect on reporter gene expression. An effect on reportergene expression is indicative of an effect on eating behavior, appetite,or the maintenance of weight. In preferred embodiments the cell is: afish cell; a reptilian cell; an amphibian cell; a mammalian cell; arodent cell, e.g., a mouse or rat cell; a primate cell; or a human cell.In preferred embodiments the cell is a neuronal cell, e.g., a GH3 cell,a PC12 cell, or a primary hypothalmic culture cell.

[0026] In another aspect, the invention features a method of evaluatinga treatment, e.g., the administration of an agent, for its effect oneating behavior, appetite, or the maintenance of weight. The methodincludes: providing an animal, cell (an animal, plant, or bacterialcell), or cell culture preparation, which expresses a receptor which isbound by MCH or which otherwise undergoes a change in its ability tobind a ligand when MCH is applied to the animal or cell, e.g., the MSHreceptor, MC3-R; administering the treatment to the animal, cell, orcell culture; and determining (1) if there is a change in a parameterrelated to binding of a ligand, e.g., an MCH agonist or antagonist, tothe receptor or (2) if there is an effect on a parameter related toeating, appetite, or weight loss or gain. In preferred embodiments thecell is a human cell transformed with a heterologus receptor, e.g., themouse receptor, e.g., a cell from the HEK-293 line or a similar cell. Inpreferred embodiments the parameter related to ligand/receptor bindingincludes: a change in a signal transduction-related phenomenon; a changein an interaction, e.g., binding, of a second ligand with the receptor,e.g., a change in the binding of ACTH ligand to the receptor. In otherpreferred embodiments the cell is: a fish cell; a reptilian cell; anamphibian cell; a mammalian cell; a rodent cell, e.g., a mouse or ratcell; a primate cell; or a human cell. In preferred embodiments the cellis a neuronal cell, e.g., a GH3 cell, a PC12 cell, or a primaryhypothalmic culture cell.

[0027] In another aspect, the invention features a method of evaluatinga treatment, e.g., the administration of an agent, for its effect oneating behavior, appetite, or the maintenance of weight. The methodincludes: providing a substrate (e.g., a substrate derived from avertebrate brain, e.g., a brain section or a synaptosome preparation) towhich MCH binds; contacting the substrate, MCH, and the agent; andevaluating the ability of the compound to promote or inhibit binding ofMCH to the substrate. The ability of the compound to inhibit MCH bindingto the substrate can be indicative of MCH agonist or antagonistactivity. In preferred embodiments: the agent is other than an antibody;the agent is other than an antibody directed against salmon MCH; theagent is other than a rabbit antibody; the agent is other than a rabbitpolyclonal antibody, e.g., other than a rabbit polyclonal anti-fish MCHantibody; the agent is other than a full length antibody, e.g., it is afragment of an antibody, e.g., a fragment capable of binding MCH; theMCH polypeptide is in a form other than a crude brain preparation orbrain slice; the MCH is substantially free of at least one protein withwhich it occurs naturally. In other preferred embodiments: the agent isa monoclonal antibody; the agent is is a recombinant or humanizedantibody.

[0028] In another aspect, the invention features a method of evaluatinga treatment, e.g., the administration of an agent, for its effect oneating behavior, appetite, or the maintenance of weight. The methodincludes: providing a cell, or cell culture preparation; administeringthe treatment; and determining if there is an effect on MCH RNA orprotein levels in the cell, or cell culture preparation. In preferredembodiments the cell is: a fish cell; a reptilian cell; an amphibiancell; a mammalian cell; a rodent cell, e.g., a mouse or rat cell; aprimate cell; or a human cell. In preferred embodiments the cell is aneuronal cell, e.g., a GH3 cell, a PC12 cell, or primary hypothalmicculture cell.

[0029] In another aspect, the invention features a method of evaluatinga treatment, e.g., the administration of an agent, for its effect oneating behavior, appetite, or the maintenance of weight. The methodincludes: providing a subject animal; administering the treatment; anddetermining if there is an effect on MCH RNA or protein levels in theanimal. The treatment can be any treatment which can result in thedesired effect but the administration of agents, e.g., drugs orchemicals, is preferred. Preferably, the treatment is other thansurgical intervention, e.g., the production of surgical lesions, e.g.,electrolytic lesions, e.g., the treatment is other than a ventromedialhypothalmic lesion, e.g., an electrolytically produced ventromedialhypothalmic lesion.

[0030] In preferred embodiments, the animal is a mammal, e.g. a rodent,e.g., a rat or mouse, a dog, or a nonhuman primate. In otherembodiments, the animal is other than a rat or mouse.

[0031] In preferred embodiments the treatment includes administering anagent and: the agent is other than an antibody; the agent is other thanan antibody directed against salmon MCH; the agent is other than arabbit antibody; the agent is other than a rabbit polyclonal antibody,e.g., other than a rabbit polyclonal anti-fish MCH antibody; the agentis other than a full length antibody, e.g., it is a fragment of anantibody, e.g., a fragment capable of binding MCH; the MCH polypeptideis in a form other than a crude brain preparation or brain slice; theMCH is substantially free of at least one protein with which it occursnaturally. In other preferred embodiments: the agent is a monoclonalantibody; the agent is a recombinant or humanized antibody.

[0032] In another aspect, the invention features a method of evaluatingan agent, e.g., a polypeptide or peptide-mimetic, for its effect oneating behavior, appetite, or the maintenance of weight The methodincludes: administering the agent to an animal, e.g., a mammal, e.g., arodent, e.g., a rat; and determining if there is an effect on aparameter related to eating behavior. In preferred embodiments, theagent is MCH, or agonist or antagonist thereof. For example, the agonistor antagonist is a peptide analog of MCH having 40, 50, 60, 70, 80, or90% homology to the native MCH, or it is a polypeptide which binds toMCH or a naturally occurring ligand of MCH, e.g., an MCH receptor, e.g.,MC3-R. The agent can be a linear or cyclic polypeptide. In preferredembodiments the compound is other than water and NaCl.

[0033] In preferred embodiments: the agent is other than an antibody;the agent is other than an antibody directed against salmon MCH; theagent is other than a rabbit antibody; the agent is other than a rabbitpolyclonal antibody, e.g., other than a rabbit polyclonal anti-fish MCHantibody; the agent is other than a full length antibody, e.g., it is afragment of an antibody, e.g., a fragment capable of binding MCH; theMCH polypeptide is in a form other than a crude brain preparation orbrain slice; the MCH is substantially free of at least one protein withwhich it occurs naturally. In other preferred embodiments: the agent isa monoclonal antibody; the agent is a recombinant or humanized antibody.In another aspect, the invention features, a method of evaluating anagent for the ability to bind an MCH polypeptide. The method includes:contacting the agent with the MCH polypeptide, or a purified preparationthereof; and evaluating ability of the compound to form a complex withthe MCH polypeptide. This method can be performed in vitro, or in vivo,e.g., in a two-hybrid interaction trap assay. In preferred embodiments;the agent is other than an antibody directed against salmon MCH; theagent is other than a rabbit antibody; the agent is other than a rabbitpolyclonal antibody, e.g., other than a rabbit polyclonal anti-fish MCHantibody; the agent is other than a full length antibody, e.g., it is afragment of an antibody, e.g., a fragment capable of binding MCH; theMCH polypeptide is in a form other than a crude brain preparation orbrain slice; the MCH is substantially free of at least one protein withwhich it occurs naturally. In other preferred embodiments: the agent isa monoclonal antibody; the agent is a recombinant or humanized antibody.

[0034] In another aspect, the invention features, a method of evaluatingan agent, e.g., a fragment of an MCH peptide, for the ability to bind,or to alter, a naturally occurring ligand of MCH, e.g., an MCH receptor,e.g., MC3-R Alter includes, e.g., sterically altering the receptor, oraltering the binding properties of the receptor for an MCH polypeptideor for another ligand. The method includes: contacting the agent withthe MCH ligand; and evaluating the ability of the agent to form acomplex with the MCH ligand, e.g., the ability of the agent to inhibitMCH peptide/MCH ligand interaction, or to alter the receptor. Thismethod can be performed in vitro, or in vivo, e.g., in a two-hybridinteraction trap assay. In preferred embodiments: the receptor is otherthan mouse MC3-R; the agent is a peptide analog of MCH having 40, 50,60, 70, 80, 90% or more homology with MCH; the agent is a linear orcyclic polypeptide.

[0035] In yet another aspect, the invention features a method forevaluating an agent, e.g., for the ability to modulate an interaction ofan MCH peptide with a second polypeptide, e.g., a naturally occurringligand of MCH, e.g., an MCH receptor, e.g., MC3-R. The method includesthe steps of (i) combining a second polypeptide (or preferably apurified preparation thereof), an MCH polypeptide (or preferably apurified preparation thereof), and the agent, e.g., under conditionswherein in the absence of the agent, the second polypeptide, and the MCHpolypeptide are able to interact, e.g., to form a complex; and (ii)detecting the interaction, e.g., detecting the formation (ordissolution) of a complex which includes the second polypeptide, and theMCH peptide. A change, e.g., a decrease or increase, in the formation ofthe complex in the presence of the agent (relative to what is seen inthe absence of the agent) is indicative of a modulation, e.g., aninhibition or promotion, of the interaction between the secondpolypeptide, and the MCH peptide. In preferred embodiments: the secondpolypeptide, and the MCH peptide, are combined in a cell-free system andcontacted with the agent; the cell-free system is selected from a groupconsisting of a cell lysate and a reconstituted protein mixture; the MCHpeptide and the second polypeptide are simultaneously expressed in acell, and the cell is contacted with the agent, e.g., the methodincludes an interaction trap assay (e.g., a two-hybrid assay). Inpreferred embodiments: the receptor is other than mouse MC3-R; the agentis a peptide analog of MCH having 40, 50, 60, 70, 80, 90% or morehomology with MCH; the agent is a linear or cyclic polypeptide.

[0036] In preferred embodiments; the agent is other than an antibodydirected against salmon MCH; the agent is other than a rabbit antibody;the agent is other than a rabbit polyclonal antibody, e.g., other than arabbit polyclonal anti-fish MCH antibody; the agent is other than a fulllength antibody, e.g., it is a fragment of an antibody, e.g., a fragmentcapable of binding MCH; the MCH polypeptide is in a form other than acrude brain preparation or brain slice; the MCH is substantially free ofat least one protein with which it occurs naturally. In other preferredembodiments: the agent is a monoclonal antibody; the agent is arecombinant or humanized antibody.

[0037] In yet another aspect, the invention features a two-phase method(e.g., a method having an in vitro and an in vivo phase) for evaluatingan agent, e.g., for the ability to modulate, e.g., to inhibit orpromote, an interaction of an MCH peptide with a naturally occurringligand of MCH, e.g., an MCH receptor, e.g., MC3-R The method includessteps (i) and (ii) of the method described immediately above performedin vitro, and further includes: (iii) determining if the agent modulatesthe interaction in vitro and if so; (iv) administering the agent to acell or animal; and (v) evaluating the in vivo effect of the agent on aninteraction, e.g., inhibition, of an MCH peptide with a secondpolypeptide, e.g., by the effect on eating behavior.

[0038] In another aspect, the invention features a two-phase method(e.g., a method having a primary in vitro and a secondary in vivo phase)for evaluating a treatment. The method can be used to evaluate atreatment for the ability to modulate, e.g., to inhibit or promote, anMCH-mediated phenomenon, e.g., an aspect of feeding behavior, appetite,or the maintenance of weight, or to evaluate a test agent for use as atherapeutic agent. The method includes: (i) an in vitro phase in whichthe test agent is contacted with a cell, or a cell free system, whichincludes a reporter gene functionally linked to an MCH regulatorysequence, and detecting the modulation of the expression of the reportergene and (ii) if the test agent modulates the expression, administeringthe test compound to an animal, and evaluating the in vivo effects ofthe compound on an aspect of feeding behavior, e.g., the level of MCHexpression.

[0039] In another aspect, the invention features, a method of evaluatingan agent for the ability to bind a nucleic acid encoding an MCHregulatory sequence. The method includes: contacting the agent with thenucleic acid; and evaluating ability of the compound to form a complexwith the nucleic acid.

[0040] In another aspect, the invention features a method of evaluatingan effect of a treatment, e.g., a treatment for treating a disordercharacterized by unwanted eating behavior, or a condition of under oroverweight. The method uses a test cell or organism which misexpressesan MCH gene. The method includes: administering the treatment to a testcell or organism, e.g., a cultured cell, or a mammal, and evaluating theeffect of the treatment on an aspect of MCH metabolism. An effect on anaspect of MCH metabolism indicates an effect of the treatment. Inpreferred embodiments: the effect on an aspect of MCH metabolism is achange in eating behavior or weight, a change in MCH mRNA levels, achange in MCH protein levels.

[0041] In preferred embodiments the cell is: a fish cell; a reptiliancell; an amphibian cell; a mammalian cell; a rodent cell, e.g., a mouseor rat cell; a primate cell; or a human cell. In preferred embodimentsthe cell is a neuronal cell, e.g., a GH3 cell, a PC12 cell, or primaryhypothalmic culture cell.

[0042] In preferred embodiments the treatment is the administration ofan agent and: the agent is other than an antibody directed againstsalmon MCH; the agent is other than a rabbit antibody; the agent isother than a rabbit polyclonal antibody, e.g., other than a rabbitpolyclonal anti-fish MCH antibody; the agent is other than a full lengthantibody, e.g., it is a fragment of an antibody, e.g., a fragmentcapable of binding MCH; the MCH polypeptide is in a form other than acrude brain preparation or brain slice; the MCH is substantially free ofat least one protein with which it occurs naturally. In other preferredembodiments: the agent is a monoclonal antibody; the agent is arecombinant or humanized antibody.

[0043] In another aspect, the invention features a method of evaluatingan effect of a treatment, e.g., a treatment for treating a disordercharacterized by unwanted eating behavior, or a condition of under oroverweight. The method uses a test cell or organism which includes MCHtransgene. The method includes: administering the treatment to a testcell or organism, e.g., a cultured cell, or a mammal, and evaluating theeffect of the treatment on an aspect of MCH metabolism. An effect on anaspect of MCH metabolism indicates an effect of the treatment. Inpreferred embodiments: the effect on an aspect of MCH metabolism is achange in eating behavior or weight, a change in MCH mRNA levels, achange in MCH protein levels. The test cell or organism can be wild typeor mutant at one or more loci other than MCH, e.g., ob, or ob receptor.The subject can also be deficient for brown fat tissue. E.g., a brownfat tissue “knockout” mouse can be made by fusing diphtheria toxin to abrown fat-specific promoter.

[0044] In preferred embodiments the cell is: a fish cell; a reptiliancell; an amphibian cell; a mammalian cell; a rodent cell, e.g., a mouseor rat cell; a primate cell; or a human cell. In preferred embodimentsthe cell is a neuronal cell, e.g., a GH3 cell, a PC12 cell, or primaryhypothalmic culture cell.

[0045] In preferred embodiments the treatment is the administration ofan agent and: the agent is other than an antibody directed againstsalmon MCH; the agent is other than a rabbit antibody; the agent isother than a rabbit polyclonal antibody, e.g., other than a rabbitpolyclonal anti-fish MCH antibody; the agent is other than a full lengthantibody, e.g., it is a fragment of an antibody, e.g., a fragmentcapable of binding MCH; the MCH polypeptide is in a form other than acrude brain preparation or brain slice; the MCH is substantially free ofat least one protein with which it occurs naturally. In other preferredembodiments: the agent is a monoclonal antibody; the agent is arecombinant or humanized antibody.

[0046] In another aspect, the invention features a method of evaluatingan effect of a treatment, e.g., a treatment for treating a disordercharacterized by unwanted eating behavior, or a condition of under oroverweight. The method uses a test cell or organism which expresses awild-type MCH gene. The method includes: administering the treatment toa test cell or organism, e.g., a cultured cell, or a mammal, andevaluating the effect of the treatment on an aspect of MCH metabolism.An effect on an aspect of MCH metabolism indicates an effect of thetreatment. In preferred embodiments: the effect on an aspect of MCHmetabolism is a change in eating behavior or weight, a change in MCHmRNA levels, a change in MCH protein levels. The test cell or organismcan be wild type or mutant at one or more loci other than MCH, e.g., ob,or ob receptor. The subject can also be deficient for brown fat tissue.E.g., a brown fat tissue “knockout” mouse can be made by fusingdiphtheria toxin to a brown fat-specific promoter.

[0047] In preferred embodiments the cell is: a fish cell; a reptiliancell; an amphibian cell; a mammalian cell; a rodent cell, e.g., a mouseor rat cell; a primate cell; or a human cell. In preferred embodimentsthe cell is a neuronal cell, e.g., a GH3 cell, a PC12 cell, or primaryhypothalmic culture cell.

[0048] In preferred embodiments the treatment is the administration ofan agent and: the agent is other than an antibody directed againstsalmon MCH; the agent is other than a rabbit antibody; the agent isother than a rabbit polyclonal antibody, e.g., other than a rabbitpolyclonal anti-fish MCH antibody; the agent is other than a full lengthantibody, e.g., it is a fragment of an antibody, e.g., a fragmentcapable of binding MCH; the MCH polypeptide is in a form other than acrude brain preparation or brain slice; the MCH is substantially free ofat least one protein with which it occurs naturally. In other preferredembodiments: the agent is a monoclonal antibody; the agent is arecombinant or humanized antibody.

[0049] In another aspect, the invention provides, a method ofdetermining if a subject mammal, e.g., a primate, e.g., a human, is atrisk for an MCH related disorder, a weight-related disorder, or aneating or appetite disorder. In preferred embodiments the method is usedto evaluate whether the subject is at risk for a genetically conditioneddisorder. Eating disorders include, e.g., a disorder characterized byunwanted eating behavior. The method includes detecting, in a tissue ofthe subject, the presence or absence of a mutation of an MCH gene. Inpreferred embodiments: detecting the mutation includes ascertaining theexistence of at least one of: a deletion of one or more nucleotides fromthe MCH gene; an insertion of one or more nucleotides into the gene, apoint mutation, e.g., a substitution of one or more nucleotides of thegene, a gross chromosomal rearrangement of the gene, e.g., atranslocation, inversion, or deletion.

[0050] For example, detecting the genetic lesion can include: (i)providing a probe/primer including an oligonucleotide containing aregion of nucleotide sequence which hybridizes to a sense or antisensesequence from MCH gene or naturally occurring mutants thereof or 5′ or3′ flanking sequences naturally associated with the MCH gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and (iii)detecting, by hybridization of the probe/primer to the nucleic acid, thepresence or absence of the genetic lesion.

[0051] Circulating white cells can be used as source of genomic DNA inthe diagnostic methods described herein. Prior art methods, e.g., thesingle strand conformation polymorphism (SSCP) method, can be used todetect lesions or polymorphisms. The diagnostic methods used herein canbe used to screen overweight subjects, e.g., obese, or morbidly obesesubjects, for MCH gene lesions or polymorphisms.

[0052] In preferred embodiments the method further includes determiningif the subject is overweight, obese, or morbidly obese. In preferredembodiments the subject is overwieght, obese, or morbidly obese.

[0053] In another aspect, the invention provides, a method ofdetermining if a subject mammal, e.g., a primate, e.g., a human, is atrisk for an MCH related disorder, a weight related disorder, or aneating disorder. In preferred embodiments the method is used to evaluatewhether the subject is at risk for a genetically conditioned disorder.The method includes detecting, in a tissue of the subject, the non wildtype levels of MCH RNA or protein. In preferred embodiments the methodfurther includes determining if the subject is overweight, obese, ormorbidly obese. In preferred embodiments the subject is overwieght,obese, or morbidly obese.

[0054] In another aspect, the invention provides, a method ofdetermining if a subject mammal, e.g., a primate, e.g., a human, is atrisk for an MCH related disorder, a weight related disorder, or aneating disorder. In preferred embodiments the method is used to evaluatewhether the subject is at risk for a genetically conditioned disorderThe method includes detecting, in a tissue of the subject, themis-expression of a gene encoding an MCH peptide. In preferredembodiments: detecting the misexpression includes ascertaining theexistence of at least one of: an alteration in the level of a messengerRNA transcript of the gene; the presence of a non-wild type splicingpattern of a messenger RNA transcript of the gene; or a non-wild typelevel of the protein. In preferred embodiments the method furtherincludes determining if the subject is overweight, obese, or morbidlyobese. In preferred embodiments the subject is overweight, obese, ormorbidly obese.

[0055] In another aspect, the invention features a method of making anMCH polypeptide, e.g., a MCH polypeptide having a non-wild typeactivity, e.g., an antagonist, agonist or super agonist of a naturallyoccurring MCH. The method includes: altering the sequence or ringstructure of an MCH peptide, preferably a mammalian, e.g., a human orrat peptide, or a peptide other than a fish, amphibian or reptilianpeptide, and testing the altered peptide for the desired activity, e.g.,by administering it to an animal and determining its effect on MCH RNAor protein levels, eating behavior or weight.

[0056] In another aspect, the invention features a cell or purifiedpreparation of cells which include a MCH transgene or which misexpressthe MCH gene. The cell preparation can consist of human or non humancells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, orpig cells. In preferred embodiments, the cell or cells include a MCHtransgene, e.g., a heterologous form of the MCH gene, e.g. a genederived from humans (in the case of a non-human cell). In otherpreferred embodiments, the cell or cells include a gene which misexpressan MCH gene, e.g., an endogenous MCH gene. In preferred embodiments, MCHis over or under expressed. Such cells can serve as a model for studyingdisorders which are related to mutated or mis-expressed MCH alleles orfor use in drug screening.

[0057] In another aspect, invention features, a transgenic MCH non-humananimal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, or a pig. Inpreferred embodiments, the transgenic animal includes (and preferablyexpress) a heterologous form of the MCH gene, e.g., a gene derived fromhumans. In other preferred embodiments, the animal has an MCH gene,e.g., an endogenous MCH gene which is misexpressed, e.g., a knockout oran overexpressed MCH gene. Such a transgenic animal can serve as a modelfor studying disorders which are related to mutated or mis-expressed MCHalleles or for use in drug screening.

[0058] For example, the invention includes a method of evaluating theeffect of the expression or misexpression of a MCH gene on a parameterrelated to eating behavior. The method includes: providing a transgenicanimal having a MCH transgene; contacting the animal with an agent,e.g., an analog of MCH; and evaluating the effect of the transgene onthe parameter (e.g., by comparing the value of the parameter for atransgenic animal with the value for a control, e.g., a wild typeanimal).

[0059] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are described in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0060] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

[0061] The drawings are briefly described.

[0062] Drawings:

[0063]FIG. 1 is a bar graph depicting MCH levels in fed versus fastedmice (after 24 hr of fasting) of the following genotype: C57/BL/6J (wildtype), ob/+, and ob/ob.

[0064]FIG. 2 is a graph depicting doubling of Kcal consumed within 1hour in rats injected intraventricularly with 5 μg of MCH.

DEFINITIONS

[0065] MCH antagonist, as used herein, refers to agents which result inan inhibition of feeding behavior. Antagonists include agents withsignificant amino acid homology to MCH as well as agents which areunrelated by amino acid sequence homology or which are not polypeptides.Antagonists include agents which act by competitively ornon-competitively binding an MCH receptor but can also include agentswhich act downstream from the receptor, e.g., in intacellular signaling,or independent of the MCH receptor. Antagonist includes “action”antagonists, e.g., agents which act by a partly or entirely differentpathway to affect eating behavior.

[0066] Misexpression, as used herein, refers to a non-wild type patternof gene expression. It includes: expression at non-wild type levels,i.e., over or under expression; a pattern of expression that differsfrom wild type in terms of the time or stage at which the gene isexpressed, e.g., increased or decreased expression (as compared withwild type) at a predetermined developmental period or stage; a patternof expression that differs from wild type in terms of decreasedexpression (as compared with wild type) in a predetermined cell type ortissue type; a pattern of expression that differs from wild type interms of the splicing size, amino acid sequence, post-transitionalmodification, or biological activity of the expressed polypeptide; apattern of expression that differs from wild type in terms of the effectof an environmental stimulus or extracellular stimulus on expression ofthe gene, e.g., a pattern of increased or decreased expression (ascompared with wild type) in the presence of an increase or decrease inthe strength of the stimulus.

[0067] A substantially pure nucleic acid, e.g., a substantially pureDNA, is a nucleic acid which is one or both of: not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (i.e., one at the 5′ end and one at the 3′ end)in the naturally-occurring genome of the organism from which the nucleicacid is derived; or which is substantially free of a nucleic acidsequence with which it occurs in the organism from which the nucleicacid is derived. The term includes, for example, a recombinant DNA whichis incorporated into a vector, e.g., into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment)independent of other DNA sequences. Substantially pure DNA also includesa recombinant DNA which is part of a hybrid gene encoding additional MCHsequence.

[0068] A purified preparation or a substantially pure preparation of apolypeptide, as used herein, means a polypeptide that has been separatedfrom other proteins, lipids, and nucleic acids with which it naturallyoccurs. Preferably, the polypeptide is also separated from substances,e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used topurify it. Preferably, the polypeptide constitutes at least 10, 20, 5070, 80 or 95% dry weight of the purified preparation. Preferably, thepreparation contains: sufficient polypeptide to allow proteinsequencing; at least 1, 10, or 100 μg of the polypeptide; at least 1,10, or 100 mg of the polypeptide.

[0069] Preparation of Peptide Analogs of MCH

[0070] Synthetic MCH and its analogs can be prepared to identifyagonists and antagonists, and determine the structural requirements forMCH agonist or antagonist activity. MCH can be modified in a number ofways, e.g., by shortening either (or both) the amino- orcarboxy-terminal regions, contracting the cysteine bridged ring, formingacyclic analogs, or modifying or substituting an amino acid, e.g., aresidue, within, or outside, the ring. Synthetic MCH and its analogs canbe assayed using one or more of the assays described herein.

[0071] Generally, the synthetic schemes use the Merrifield solid phasesynthesis followed by cyclization and purification as described, e.g.,in Lebl et al. (1988) J. Med. Chem. 31:949-954, herein incorporated byreference. Briefly, chloromethylated resin can be used as the support tointroduce the first amino acid on an automated synthesizer, e.g. aDuPont 2200. The intact peptides are cleaved from the resin and thenwashed. Following extraction from the wash the peptides are lyophilized.The lyophilized protein is dissolved in degassed water. Cyclization isachieved by the dropwise addition of potassium ferricyanide (K₃Fe(CN)₆).Purification can be performed by column chromatography on Sephadex G-25,carboxymethyl cellulose and by reversed-phase high-performancechromatography (HPLC).

[0072] Alternatively, truncated MCH analogs can be prepared by exposingnatural or synthetic MCH to enzymes. Natural MCH can be isolated frompituitaries using an acetone extraction and purified on an HPLC columnas described in Kawauchi et al. (1988) Adv. in Pigment Cell Res.517-530, herein incorporated by reference. For example, MCH₁₋₁₄, acarboxy-terminal truncation, can be generated from MCH by exposure tocarboxypeptidase Y.

[0073] Acyclic analogs can be constructed by replacing the Cys⁵-Cys¹⁴bridge with pseudoisosteric residues. Either L-serine, a polarsubstitute, or L-α-aminobutyrate, a non-polar substitute, can beutilized. The peptides, with the appropriate substitution, can beprepared by solid phase synthesis as described above and in Matsunaga etal., Life Sci. (1992) 51:679-685, herein incorporated by reference.

[0074] Modification of amino acids within the ring is performed with areagent specific to each residue. Modifications can be accomplishedeither by substituting a different amino acid or altering the existingamino acid. For example, the Tyr residue at position 11 can be modifiedwith the addition of a —NO₂ group by exposing MCH to a solution of 10%nitromethane-95% ethanol. See, e.g., Kawauchi et al. (1988) Adv. inPigment Cell Res. 517-530, herein incorporated by reference.

[0075] Assays for MCH Agonist and Antagonist Activity

[0076] Frog/Lizard Melanocyte Assay

[0077] The activity of compounds, e.g., MCH and related analogs, e.g.,MCH agonists or antagonists, can be determined by in vitro assay usingskins from frogs (R. pipiens) and lizards (A. carolinensis) (e.g., seeCastrucci et al. (1989) Gen. Comp. Endocrinol. 73:157-163, and Hruby etal. (1987) J. Med. Chem. 30:2126-2130, herein incorporated byreference).

[0078] These assays are based upon the amount of light reflected fromthe surface of the skins in vitro. In these assays, melanin granules(melanosomes) within melanocytes migrate outward into the dendriticprocesses of the pigment cells in response to MCH. This centrifugalgranule translocation results in darkening of the skins. The changes inreflectance are measured by a reflectometer and are usually expressed asa percent change from the initial base (zero time) values. An increasein reflectance indicates skin lightening whereas a decrease inreflectance indicates skin darkening. Removal of the MCH (i.e., byrinsing with Ringer solution) from the incubation medium usually resultsin a perinuclear aggregation of melanosomes leading to a lightening ofthe skins back to original base values.

[0079] Briefly, frogs or lizards are sacrificed by decapitation and theleg and thigh skins are removed and kept viable in a bath ofphysiological saline (Ringer) solution. Skins can than be stretched overPVC rings and the baseline light reflectance measured by reflectometer.For example, to assay for antagonistic activity, the skins can bepre-incubated for 1 hour in various concentrations of the potentialantagonist. After this period, a known concentration of MCH can be addedand its activity determined in the same assay. Therefore, dose responsecurves for each potential MCH agonist or antagonist and the relativepotency, as compared to MCH, can be calculated.

[0080] Fish Scale Melanocyte Assay

[0081] The activity of compounds, e.g., MCH and related analogs, e.g.,agonists or antagonists, can also be evaluated in various fishscale-based melanocyte assays. In this assay fish scales are used tovisualize the actions of MCH related compounds. The scales from avariety of fishes, such as the yearling tilapia (Oreochromismossambicus), can be used. See, e.g., Hogben and Slome (1931) Proc. R.Soc. B108:10-53, herein incorporated by reference.

[0082] Briefly, the scales are incubated in a solution that dispersesthe melanin causing the scales to darken. A sample of unknown MCHactivity is added. If there is MCH-like activity the melanin isconcentrated causing the scales to lighten. Alternatively, the scalescan be incubated with a combination of MCH and putative antagonist toevaluate the antagonists potency. The scales are evaluated visuallyunder a microscope. See, e.g., Kawauchi et al., Nature, 305:321-323(1983), incorporated herein by reference.

[0083] Teleost Skin Melonocyte Bioassays

[0084] Yet another method utilizes the teleost fish, Synbranchusmarmoratus, to evaluate the melanosome aggregating activity of MCH, MCHanalogs, or a sample containing unknown MCH activity. In this assay the“resting” (unstimulated) state of the melanophores is characterized bydispersed melanosomes, i.e., the skin is darkened. Therefore, the assayis particularly appropriate for the study of melanosome aggregatingagents such as MCH. This assay can be performed as essentially disclosedin Casstucci et al., Gen. Comp. Endocrin., 66:374-380 (1987),incorporated herein by reference.

[0085] Briefly, fish skins are cut into pieces approximately 2.5×2.5 cm,placed between two rings (either PVC or metal), and allowed toequilibrate in a suitable buffered solution, preferably Tyrode's orRinger's, for an hour. In the resting (unstimulated) state the skins aredarkened. MCH is added to the bath and incubated with the fish skins for60 minutes. The changes in skin color are measured by a reflectometer.The MCH will cause the skins to lighten and will result in higherreflectance values.

[0086] In Vivo Melanocyte Bioassay

[0087] The MCH-like activity of analogs or samples can also be evaluatedin an intact organism. If rainbow trout are exposed to a blackbackground their scales will darken. Intraperitoneal injections of MCH,its analog, or a sample of unknown MCH activity into the darken troutwill result in a lightening of the scales if there is MCH-like activity.The effect is rapid in onset and lasts for several hours if there isMCH-like activity. This assay can be performed as essentially disclosedin Kawauchi et al., Adv. Pigment Cell Res., 517-530 (1988), incorporatedherein by reference.

[0088] Radioimmunoassay and Immunohistochemistry: Methods for Detectingthe Levels of MCH Binding to a Substrate

[0089] The concentration of MCH in an individual or a tissue sample canbe determined by radioimmunoassay. The sample containing an unknown MCHconcentration is compared with a standard of known concentration. As asample contains an increasing amount of MCH there will be more unlabeledMCH available for binding to the anti-MCH antibody relative toradiolabeled MCH resulting in less radiolabeled MCH being bound by theanti-MCH antibody. This can be used to determine the binding of MCH oran analog thereof to a substrate in, e.g., the presence or absence ofanother compound, e.g., a putative antagonist. Generally, a sample isobtained from the subject and protein samples prepared. Rabbit anti-MCHcan serve as the primary antibody and is incubated with the proteinsample to which a radiolabeled MCH had been added. Goat anti-rabbit isthen added to aid in precipitating the MCH-rabbit antibody complex.Following an incubation period the sample is centrifuged and theresulting pellet is counted. The more MCH there is in the sample theless radioactivity will be found in the resulting pellet. This assay canbe performed as essentially disclosed in Zamir et al., supra.

[0090] Localization of MCH can be accomplished by fluorescentimmunohistochemistry. For example, rat brains can be sectioned into 20μM thick slices and placed onto microscope slides. The slices areincubated with a rabbit anti-MCH antibody or equivalent antibody. Theslices are washed to remove excess antibody. An incubation with afluorescently labeled goat anti-rabbit antibody allows for localizationof the MCH-like material. Fluorescence can be monitored with afluorescence microscope. This assay can be performed as essentiallydisclosed in Zamir et al., Proc. Natl. Acad. Sci. USA, 83:1528-1531(1986), herein incorporated by reference.

[0091] Synaptosome Binding

[0092] The ability of a compound to bind a naturally occurring MCHreceptor, e.g., the ability of analogs to competitively antagonize MCH,can be determined utilizing radiolabeled MCH in binding assays. Acompetitive antagonist can prevent MCH from interacting with itsreceptor and less radioactivity would be bound. A highly tritiated-MCHhas been synthesized and is available for whole cell binding studies,see, e.g., Drozdz and Eberle, J. Receptor & Signal Transduction Res.15(1-4):487-502 (1995), herein incorporated by reference. Methods foriodinating MCH have also been developed, see, e.g., Drozdz and Eberle,23rd European Peptide Symphosium, Braga, Portugal (1994), herebyincorporated by reference.

[0093] Generally, the tissue sample of interest is homogenized whenpreparing membranes or synaptosomes, or digested with enzymes for wholecells. Whole cells, membranes or synaptosomes can then be isolated bycentrifugation. For example, membranes can be prepared by homogenizingthe tissue sample, and centrifuging the resultant solution. Thesupernatant is collected and centrifuged. The pellet is resuspended andpassed through a small gauge needle. The crude membrane pellet isresuspended in an appropriate binding buffer. Membranes are exposed to aconstant concentration of radiolabeled MCH and varying concentrations ofthe analog of interest. Unbound ³H-MCH is separated from the bound³H-MCH by a rapid filtration over fiber glass filters. The filters arewashed and counted. See, e.g., Drozdz and Eberle, J. Receptor & SignalTransduction Res. 15(1-4):487-502 (1995), herein incorporated byreference.

[0094] HEK-293 Assay

[0095] As discussed herein, heterologous human kidney, HEK-293 cellsstably transfected with a plasmid carrying MC3-R can be used to assayMCH activity. It is not clear whether the MCH interaction with the MC3-Rreceptor is direct or indirect, but application of MCH results in anincrease in binding of ACTH to the MC3-R receptor. Scatchered analysissuggests an increase in ACTH binding sites, so the effect of MCH may beto induce a steric change in the receptor. Exposure of cells to 10-7 MMCH for 20 minutes or overnight results in a 15-100% increase in ACTHbinding.

[0096] In Vivo Rodent Brain Assay

[0097] As discussed herein, rodents, e.g., rats, can be used to assayMCH activity in vivo. A Teflon catheter can be inserted into the thirdventricle of a rat and cemented into place. MCH or its analogs, e.g.,agonists or antagonists, can be introduced by way of the catheter atvarious concentrations, and their effect on eating behavior determined.

[0098] Identification of Genes Preferentially Expressed in ob/obHypothalamus by PCR Display

[0099] PCR display was used to identify differential expression ofneuropeptides that might be important in appetite regulation in thehypothalamus of obese rodents. PCR display allows screening fordifferential gene expression with relatively small amounts (100 ug) ofmRNA (Lian and Pardee, 1992, Science 257:967-971).

[0100] PCR display was performed as follows. Male C57b16J ob/ob, ob/+heterozygotes, and unaffected C57b16J mice were obtained at 7 weeks ofage from Jackson Laboratories (Bar Harbor, Me.). Mice were housed for atleast 4 days after arrival, to allow them to recover from shipping. Fedmice were sacrificed in the morning, after being anesthetized with an IPinjection of 200 mg/kg of sodium amytal. In small experiments food waswithdrawn at the time interval described. Mice were decapitated, thebrains were removed, and the hypothalami identified, excised, andextracted immediately in RNAzol (Cinna/Biotex Laboratories, Houston,Tex.). 10 hypothalami, weighing approximately 100 mg, were collected andthen homogenized using a hand held homogenizer.

[0101] Aliquots of total RNA were treated with DNAase I (BoehringerMannheim, Indianapolis, Ind.) to remove any traces of DNA. RNA wasdivided into nine pools, and a cDNA was synthesized using MMTV reversetranscriptase (Superscript RNAaseH, Gibco BRL, Gaithersburg, Md.), andone of nine anchored primers (see below). cDNA, thus generated, was usedin PCR display.

[0102] 12 possible downstream primers with the sequence T₁₂XY (where Xand Y are any nucleotide) and termed anchored primers, were used inconjunction with approximately 50 upstream random primers, designatedarbitrary primers for PCR display. The arbitrary upstream primers didnot contain more than 50% GC and had no internal homology to each other.Using these 600 primer pairs, it was possible to assess expression ofabout 30,000 mRNAs (each primer pair yielded approximately 50 cDNAbands). In the present study, 180 primers pairs consisting of one ofnine anchored primers and 20 arbitrary primers were used to screen mRNAexpression in hypothalamus of obese vs. non-obese animals. cDNAgenerated from the reverse transcriptase reaction was amplified usingAmplitaq DNA polymerase (Perkin Elmer, Norwalk Conn.). Reactions wereperformed in the presence of ³⁵S-ATP (NEN, Boston, Mass.), and productswere separated on sequencing gels. Dried gels were exposed to KodakX-OMAT AR film (Eastman Kodak, Rochester, N.Y.) for 24 to 48 hours.After development, DNA fragments from ob/ob and ob/+ hypothalamus werecompared.

[0103] Analysis of ob/ob Expressed Genes

[0104] 52 DNA bands appeared to be differentially expressed on the PCRdisplay reaction. 35 of these 52 bands were evaluated using Northernblot analysis with riboprobes. Of these no signal could be detected for9 bands, and no difference in expression was observed in 20 bands. Thus,of about 9,000 cDNAs screened, differences in expression were confirmedfor only six bands (or about 0.7%). Of these, two had matches in GeneBank: one was melanin concentrating hormone (MCH) and the other, themouse oncogene, fau. A third band had homology to a DNA binding factor,and three additional bands which were differentially expressed had noknown homology. Although the difference in MCH expression ondifferential display appeared to be absolute, i.e. no signal wasdetected in the ob/+ mice versus an obvious signal in ob/ob mice,assessment of MCH expression using a ribonuclease protection assayshowed that the difference between fed ob/ob and ob/+ mice was a rathermodest 50-80% increase in the ob/ob animals.

[0105] Differentially expressed bands were identified as follows. Bandsunique to either ob/ob or ob/+ hypothalamus were excised from the driedgel and were extracted by boiling 100 μL of TE buffer and precipitatedwith ethanol in the presence of muscle glycogen (Boehringer Mannheim,Indianapolis, Ind.). DNA was further amplified using the original set ofprimers used to generate the particular band, under the same thermalcycling conditions. Reaction products were run out on a 1% agarose geland stained with ethidium bromide. Bands were excised from the gel,eluted, and ligated into the pCR plasmid (InVitrogen, San Diego,Calif.).

[0106] Bands inserted into the pCR vector were sequenced usingdideoxysequencing in order to determine both sequence and orientation.Depending on orientation, the T4 or T7 promoter was utilized to generatea riboprobe. Riboprobes were used to probe Northern blots containing 20μg/lane of RNA from ob/ob or ob/+ hypothalamus. Northern blots wereexposed to either Kodak X-OMAT film or analyzed using the MolecularDynamics PhosphoImager.

[0107] Expression of MCH

[0108] To further evaluate the difference in expression of MCH in leanversus obese mice, and to evaluate the possibility that differences weresusceptible to nutritional status, control C57B16J mice, C57B16J ob/+heterozygotes and C57B16J ob/ob animals were compared both in the fedstate and after 24 hours of fasting. FIG. 1 shows quantitative dataderived from hypothalmic mRNA blots in fed and fasted mice probed withMCH. MCH expression is 233% increased in fed ob/ob mice when compared tolean mice without the ob gene. Lean heterozygotes are intermediatebetween the two mice and MCH mRNA is 156% increased over control levels.Fasting for 24 hours increased MCH expression in all three groups ofmice. Expression in control mice was increased to 233% compared tofasted animals. The relative ratio of MCH mRNA levels in control, ob/+and ob/ob mice remained the same, but the total of MCH mRNA doubled foreach group.

[0109] Levels of NPY mRNA were measured as “control” neuropeptide. Inthe fed state NPY expression was slightly higher (161%) in ob/ob mice ascompared to either control homozygote or ob/+ mice. Levels of NPY mRNArose with fasting and NPY expression (control fasted 172% of fed mice)was two fold higher in fasted ob/ob mice as compared to fed ob/ob.

[0110] The changes in MCH expression over time were also evaluated infasted C57B16J lean animals. A rise in MCH expression was detected sixhours after onset of fasting and increased through 24 hours.

[0111] Because, other investigators have reported extra-hypothalamicexpression, Northern blots loaded with 30 μg of RNA and probed with ariboprobe were used to screened a panel of organs for MCH expression. NoMCH signal could be detected except in the hypothalamus.

[0112] Administration of MCH In Vivo

[0113] To further evaluate the importance of MCH as an appetiteregulator, a Teflon catheter was inserted into the third ventricle of arat and cemented into place, followed by intraventricular injection of 5μg of MCH in 5 μl of phosphate buffered saline. Control animals receivedonly phosphate buffered saline. Administration of MCH increased feedingbehavior, more than doubling Kcal consumed within one hour (FIG. 2).

[0114] Structural Requirements for MCH Activity

[0115] A significant amount of work has been done on determiningstructural requirements for MCH-like activity in melanocyte assaysystems using analogs of salmon MCH peptide. Analogs derived from thiswork can be tested for MCH-like activity using methods of the invention,e.g., in vivo rat assay or in vitro HEK-293 receptor binding assay, ornew MCH analogs, e.g., human MCH analogs, based on the prior artknowledge on structural requirements, can be synthesized and tested foractivity in one of the in vivo or in vitro assays described herein.

[0116] Numerous investigators have synthesized N-terminal and C-terminalfragment analogs of salmon MCH and have tested them for MCH activity inteleost skin bioassay and frog and lizard bioassays, described herein(see, e.g., Matsunaga et al. (1989) Peptides 10:349-354; Hardley et al.(1987) Life Sci. 40:1139-1145, herein incorporated by reference). Thesestudies have concluded that the minimal sequence needed to elicit anequipotent response to the native MCH is MCH (5-15), a structure whichlacks residues 1-4 from the N-terminal end, and residues 16-17 of theC-terminal end of the peptide. The removal of Trp¹⁵, producing afragment MCH (5-14), results in an analog 100 to 300 less active thannative MCH indicating that Trp at position 15 is important formaintenance of full (equipotent) agonist activity of MCH, and thatindole ring of Trp residue may be important in aiding the fit of MCHinto its receptor pocket, thus facilitating binding. Because fragmentanalogs, which are N terminal deleted, e.g., those lacking residues 1-4,are equipotent to native MCH, they appear to not be required for MCHactivity. The same was concluded for residues 16-17 in the C-terminalend of the peptide.

[0117] Furthermore, other investigators have synthesized MCH analogswith contracted ring structure and have tested them for activity inteleost fish skin bioassay (see, e.g., Lebl et al. (1988) J. Med. Chem.31:949-954; Lebl et al. (1989) Life Sci. 44:451-457; Matsunaga et al.(1989) Peptides 10:349-354, herein incorporated by reference). Thefollowing ring contraction analogs (which retain a disulfide bond) weresynthesized: [Ala⁵, Cys¹⁰]MCH, [Ala⁵, Cys⁸]MCH, [Ala⁵, Cys⁷]MCH, [Ala⁵,Cys¹⁰]MCH₅₋₁₇, [Ala⁵, Cys⁸]MCH₅₋₁₇, [Ala⁵, Cys⁷]MCH₅₋₁₇,[Cys¹⁰]MCH₁₀₋₁₇, [Cys⁸]MCH₈₋₁₇, and [Cys⁷]MCH₇₋₁₇. The studies withthese analogs have concluded that the disulfide bond between positions 5and 14 is essential for the MCH-like activity, because ring contractionseliminated or greatly reduced the MCH-like activity. It seems that the10 ring residue structure, MCH (5-14) is very important for optimalactivation. Surprisingly, two of the analogs, [Ala⁵, Cys⁸]MCH₅₋₁₇ and[Cys¹⁰]MCH₁₀₋₁₇, were found to be full agonists, however, with veryreduced potency, indicating that the shortest sequence having MCH-likeactivity may be comprised of residues 10-14 (Val-Tyr-Arg-Pro-Cys) withresidues at positions 11-14 (Tyr-Arg-Pro-Cys) possibly being crucial formessage transduction.

[0118] In addition, acyclic analogs have been synthesized and tested forMCH activity in teleost fish skin bioassay (see, e.g., Kawauchi andKawazoe (1988) Advances in Pigment Cell Res. 517-527; Matsunaga et al.(1992) Life Sci. 51:679-685, herein incorporated by reference). Theseanalogs were constructed so that they differed form native MCH only inthe polarity of the side chain group at positions 5 and 14. For oneanalog polar L-serine was substituted for cysteine at positions 5 and 14(L-Ser^(5,14) MCH), while for the other analog, non-polar Lα-aminobutyrate (Abu) was substituted at the same positions (Abu^(5,14)MCH). Another acyclic analog was constructed by reduction of thedisulfide bond, followed by subsequent carboxymethylation of Cysresidues at positions 5 and 14 (CAM-Cys^(5,14) MCH). All of theseanalogs exhibited no MCH-like activity, suggesting that the disulfidebridge is necessary to maintain correct conformation and topographicalfeatures of MCH for receptor binding and transmembrane signaltransduction.

[0119] MCH derivatives with modified residues have also been synthesizedand tested for activity in fish scale assay (see, e.g., Kawauchi andKawazoe (1988) Advances in Pigment Cell Res. 517-527, hereinincorporated by reference). The following derivatives have beensynthesized and tested for activity: NPS-Trp¹⁵MCH, DHCH-Arg^(4,9,12)MCH,NO₂-Tyr¹¹MCH and S-O-Met^(3,6)MCH. Modifications of amino acid residuesoutside of the ring structure had no effect on the MCH activity, whilethe modifications of residues within the ring, e.g.,DHCH-Arg^(4,9,12)MCH, NO₂-Tyr¹¹MCH and S-O-Met^(3,6)MCH, resulted inanalogs with greatly reduced MCH activity. These results support thesuggestion that the MCH activity is elicited from the cyclic segment(MCH5-14) of the peptide.

[0120] The melanocyte-based assays predict that peptides of thefollowing structure will be useful as agonists of MCH activity:

[0121]R¹-R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸-R⁹-R¹⁰-R¹¹-R¹²-R¹³-R¹⁴-R¹⁵-R¹⁶-R¹⁷-R¹⁸-R¹⁹

[0122] wherein:

[0123] R¹ is Asp, a conserved amino acid substitution from the tableprovided herein, any D amino acid, or deleted;

[0124] R² is Phe, a conserved amino acid substitution from the tableprovided herein, any D amino acid, or deleted;

[0125] R³ is Asp, a conserved amino acid substitution from the tableprovided herein, any D amino acid, or deleted;

[0126] R⁴ is Met or a conserved amino acid substitution from the tableprovided herein, Thr or a conserved amino acid substitution from thetable provided herein, any D amino acid, or deleted;

[0127] R⁵ is Leu or a conserved amino acid substitution from the tableprovided herein, Met or a conserved amino acid substitution from thetable provided herein, any D amino acid, or deleted;

[0128] R⁶ is Arg, a conserved amino acid substitution from the tableprovided herein, any D amino acid, deleted, or Cys;

[0129] R⁷ is Cys, or any amino acid;

[0130] R⁸ is Met, a conserved amino acid substitution from the tableprovided herein, or Cys;

[0131] R⁹ is Leu or a conserved amino acid substitution from the tableprovided herein, or Val or a conserved amino acid substitution from thetable provided herein;

[0132] R¹⁰ is Gly, or a conserved amino acid substitution from the tableprovided herein;

[0133] R¹¹ is Arg, or a conserved amino acid substitution from the tableprovided herein;

[0134] R¹² is Val, or a conserved amino acid substitution from the tableprovided herein;

[0135] R¹³ is Tyr, or a conserved amino acid substitution from the tableprovided herein;

[0136] R¹⁴ is Arg, or a conserved amino acid substitution from the tableprovided herein;

[0137] R¹⁵ is Pro, a conserved amino acid substitution from the tableprovided herein, or Cys;

[0138] R¹⁶ is Cys, or any amino acid;

[0139] R¹⁷ is Trp, a conserved amino acid substitution form the tableprovided herein, an analog of Trp, e.g., NPS-Trp, an amino acid havingan aromatic side group, or Cys;

[0140] R¹⁸ is Gln or a conserved amino acid substitution from the tableprovided herein, Glu or a conserved amino acid substitution from thetable provided herein, Trp or a conserved amino acid substitution formthe table provided herein, an analog of Tip, e.g., NPS-Trp, an aminoacid having an aromatic side group, or deleted;

[0141] R¹⁹ is Val, a conserved amino acid substitution from the tableprovided herein, or deleted;

[0142] provide that: if R⁶ is Cys, then R¹⁵ is Cys, the disulfide bridgeis formed between the two, and R⁷, R⁸, R¹⁶ and R¹⁷ are not Cys; if R⁷ isCys, then R¹⁶ is Cys, the disulfide bridge is formed between the two,and R⁶, R⁸, R¹⁵ and R¹⁷ are not Cys; if R⁸ is Cys, then R¹⁷ is Cys, thedisulfide bridge is formed between the two, R⁶, R⁷, R¹⁵ and R¹⁶ are notCys, and R¹⁸ is Trp or a conserved amino acid substitution form thetable provided herein, an analog of Trp, e.g., NPS-Trp, or an amino acidhaving an aromatic side group.

[0143] In preferred embodiments: R¹² is Val, R¹³ is Tyr, R¹⁴ is Arg, R¹⁵is Pro, R¹⁶ is Cys and R¹⁷ is Trp; the agonist has a disulfide bridgebetween residues R⁷ and R¹⁶; the disulfide ring includes ten aminoacids; the agonist is deleted for any or all of the residues between R¹and R⁶; the agonist is deleted for one or both of the residues betweenR¹⁸ and R¹⁹; the agonist has at least 70, 80, or 90% homology withhuman, rat or salmon MCH; the agonist has 1, 2, 3, 4, 5 or more residueswithin the ring modified or substituted with a conserved amino acid fromthe table provided herein.

[0144] In preferred embodiments, the agonist is: MCH(2-19), MCH(3-19),MCH(4-19), MCH(5-19), MCH (6-19), MCH(7-19), MCH(1-18), MCH(2-18),MCH(3-18), MCH(4-18), MCH(5-18), MCH(6-18), MCH(7-18), MCH(1-17),MCH(2-17), MCH(3-17), MCH(4-17), MCH(5-17), MCH(6-17), MCH(7-17), andNPS-Trp¹⁷MCH.

[0145] The melanocyte-based assays predict that the peptides of thefollowing structure will be useful as antagonists of MCH activity:

[0146]R¹-R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸-R⁹-R¹⁰-R¹¹-R¹²-R¹³-R¹⁴-R¹⁵-R¹⁶-R¹⁷-R¹⁸-R¹⁹

[0147] wherein:

[0148] R¹ is Asp, a conserved amino acid substitution from the tableprovided herein, any D amino acid, or deleted;

[0149] R² is Phe, a conserved amino acid substitution from the tableprovided herein, any D amino acid, or deleted;

[0150] R³ is Asp, a conserved amino acid substitution from the tableprovided herein, any D amino acid, or deleted;

[0151] R⁴ is Met or a conserved amino acid substitution from the tableprovided herein, Thr or a conserved amino acid substitution from thetable provided herein, any D amino acid, or deleted;

[0152] R⁵ is Leu or a conserved amino acid substitution from the tableprovided herein, Met or a conserved amino acid substitution from thetable provided herein, any D amino acid, or deleted;

[0153] R⁶ is Arg, a conserved amino acid substitution from the tableprovided herein, any D amino acid, deleted, or Cys;

[0154] R⁷ is Cys, or any amino acid;

[0155] R⁸ is Met, a conserved amino acid substitution from the tableprovided herein, or Cys;

[0156] R⁹ is Leu or a conserved amino acid substitution from the tableprovided herein, or Val or a conserved amino acid substitution from thetable provided herein;

[0157] R¹⁰ is Gly, or a conserved amino acid substitution from the tableprovided herein;

[0158] R¹¹ is Arg, or a conserved amino acid substitution from the tableprovided herein;

[0159] R¹² is any amino acid other than Val, or other than a conservedamino acid replacement;

[0160] R¹³ is any amino acid other than Tyr, or other than a conservedamino acid replacement;

[0161] R¹⁴ is any amino acid other than Arg, or other than a conservedamino acid replacement;

[0162] R¹⁵ is any amino acid other than Pro, other than a conservedamino acid replacement, or Cys;

[0163] R¹⁶ is Cys, or any other amino acid;

[0164] R¹⁷ is Trp or a conserved amino acid substitution form the tableprovided herein, an analog of Trp, e.g., NPS-Trp, an amino acid havingan aromatic side group, any amino acid other than Trp, other than aconserved amino acid replacement, an amino acid lacking an aromatic sidegroup, deleted, or Cys;

[0165] R¹⁸ is Gln or a conserved amino acid substitution from the tableprovided herein, Glu or a conserved amino acid substitution from thetable provided herein, Trp or a conserved amino acid substitution formthe table provided herein, an analog of Trp, e.g., NPS-Trp, an aminoacid having an aromatic side group, any amino acid other than Trp, otherthan a conserved amino acid replacement, an amino acid lacking anaromatic side group, or deleted;

[0166] R¹⁹ is Val, a conserved amino acid substitution from the tableprovided herein, or deleted;

[0167] provided that: if R⁶ is Cys, then R¹⁵ is Cys, the disulfidebridge is formed between the two, and R⁷, R⁸, R¹⁶ and R¹⁷ are not Cys;if R⁷ is Cys, then R¹⁶ is Cys, the disulfide bridge is formed betweenthe two, and R⁶, R⁸, R¹⁵ and R¹⁷ are not Cys; if R⁸ is Cys, then R¹⁷ isCys, the disulfide bridge is formed between the two, R⁶, R⁷, R¹⁵ and R¹⁶are not Cys, and R¹⁸ is Trp or a conserved amino acid substitution formthe table provided herein, an analog of Trp, e.g., NPS-Trp, an aminoacid having an aromatic side group, any amino acid other than Trp, otherthan a conserved amino acid replacement, an amino acid lacking anaromatic side group, or deleted.

[0168] In preferred embodiments:

[0169] R¹² is any amino acid other than Val, or other than a conservedamino acid replacement;

[0170] R¹³ is any amino acid other than Tyr, or other than a conservedamino acid replacement;

[0171] R¹⁴ is any amino acid other than Arg, or other than a conservedamino acid replacement;

[0172] R¹⁵ is any amino acid other than Pro, or other than a conservedamino acid replacement;

[0173] R¹⁶ is Cys;

[0174] R¹⁷ is any amino acid other than Trp, other than a conservedamino acid replacement, an amino acid lacking an aromatic side group, ordeleted.

[0175] In preferred embodiments: the antagonist has a disulfide bridgebetween residues R⁷ and R¹⁶; the disulfide ring includes ten aminoacids; the antagonist is deleted for any or all of the residues betweenR¹ and R⁶; the antagonist is deleted for one or both of the residuesbetween R¹⁸ and R¹⁹; the antagonist has at least 70, 80, or 90% homologywith human, rat or salmon MCH; the agonist has 1, 2, 3, 4, 5 or moreresidues within the ring modified or substituted with a nonconservedamino acid.

[0176] In preferred embodiments, the antagonist is: MCH(1-16),MCH(2-16), MCH(3-16), MCH(4-16), MCH(5-16), MCH(6-16), MCH(7-16),DHCH-Arg^(6,11,14)MCH, and NO₂-Tyr¹³MCH.

[0177] Transgenic Animals

[0178] The invention includes transgenic animals which include cells (ofthat animal) which contain an MHC transgene and which preferably express(or misexpress) an endogenous or exogenous MCH in one or more cells inthe animal. The MCH transgene can encode the wild-type form of theprotein, or can encode homologs thereof, including both agonists andantagonists, as well as antisense constructs. In preferred embodiments,the expression of the transgene is restricted to specific subsets ofcells, or tissues, e.g., hypothalamus, utilizing, for example,cis-acting sequences that control expression in the desired pattern.Tissue-specific regulatory sequences and conditional regulatorysequences can be used to control expression of the transgene in certainspatial patterns. Temporal patterns of expression can be provided by,for example, conditional recombination systems or prokaryotictranscriptional regulatory sequences.

[0179] Genetic techniques which allow for the expression of transgenes,that are regulated in vivo via site-specific genetic manipulation, areknown to those skilled in the art. For example, genetic systems areavailable which allow for the regulated expression of a recombinase thatcatalyzes the genetic recombination a target sequence. As used herein,the phrase “target sequence” refers to a nucleotide sequence that isgenetically recombined by a recombinase. The target sequence is flankedby recombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of the subject MCH polypeptide. For example, excision of atarget sequence which interferes with the expression of a recombinantMCH gene, such as one which encodes an antagonistic homolog, can bedesigned to activate expression of that gene. This interference withexpression of the protein can result from a variety of mechanisms, suchas spatial separation of the MCH gene from the promoter element or aninternal stop codon. Moreover, the transgene can be made wherein thecoding sequence of the gene is flanked recombinase recognition sequencesand is initially transfected into cells in a 3′ to 5′ orientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

[0180] See e.g., descriptions of the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCTpublication WO 92/15694).

[0181] Genetic recombination of the target sequence is dependent onexpression of the Cre recombinase. Expression of the recombinase can beregulated by promoter elements which are subject to regulatory control,e.g., tissue-specific, developmental stage-specific, inducible orrepressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of the recombinant MCH can be regulated viacontrol of recombinase expression.

[0182] Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080. Moreover, expression of theconditional transgenes can be induced by gene therapy-like methodswherein a gene encoding the trans-activating protein, e.g. a recombinaseor a prokaryotic protein, is delivered to the tissue and caused to beexpressed, such as in a cell-type specific manner. By this method, theMCH transgene could remain silent into adulthood until “turned on” bythe introduction of the trans-activator.

[0183] Gene Therapy

[0184] The gene constructs of the invention can also be used as a partof a gene therapy protocol to deliver nucleic acids encoding either anagonistic or antagonistic form of an MCH peptide. The invention featuresexpression vectors for in vivo transfection and expression of an MCHpeptide in particular cell types so as to reconstitute the function of,or alternatively, antagonize the function of MCH peptide in a cell inwhich that peptide is misexpressed. Expression constructs of MCHpeptides, may be administered in any biologically effective carrier,e.g. any formulation or composition capable of effectively deliveringthe MCH gene to cells in vivo. Approaches include insertion of thesubject gene in viral vectors including recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly; plasmid DNA can be delivered with the help of, forexample, cationic liposomes (lipofectin) or derivatized (e.g. antibodyconjugated), polylysine conjugates, gramacidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo.

[0185] A preferred approach for in vivo introduction of nucleic acidinto a cell is by use of a viral vector containing nucleic acid, e.g. acDNA, encoding an MCH polypeptide. Infection of cells with a viralvector has the advantage that a large proportion of the targeted cellscan receive the nucleic acid. Additionally, molecules encoded within theviral vector, e.g., by a cDNA contained in the viral vector, areexpressed efficiently in cells which have taken up viral vector nucleicacid.

[0186] Retrovirus vectors and adeno-associated virus vectors can be usedas a recombinant gene delivery system for the transfer of exogenousgenes in vivo, particularly into humans. These vectors provide efficientdelivery of genes into cells, and the transferred nucleic acids arestably integrated into the chromosomal DNA of the host. The developmentof specialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). A replication defectiveretrovirus can be packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad.Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

[0187] Another viral gene delivery system useful in the presentinvention utilizes adenovirus-derived vectors. The genome of anadenovirus can be manipulated such that it encodes and expresses a geneproduct of interest but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle. See, for example, Berkneret al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 d1324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they are not capable of infectingnondividing cells and can be used to infect a wide variety of celltypes, including epithelial cells (Rosenfeld et al. (1992) cited supra).Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267).

[0188] Yet another viral vector system useful for delivery of thesubject MCH gene is the adeno-associated virus (AAV). Adeno-associatedvirus is a naturally occurring defective virus that requires anothervirus, such as an adenovirus or a herpes virus, as a helper virus forefficient replication and a productive life cycle. (For a review seeMuzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129).It is also one of the few viruses that may integrate its DNA intonon-dividing cells, and exhibits a high frequency of stable integration(see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlinet al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as300 base pairs of AAV can be packaged and can integrate. Space forexogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

[0189] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of anMCH peptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject MCH gene by thetargeted cell. Exemplary gene delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

[0190] In a representative embodiment, a gene encoding an MCHpolypeptide can be entrapped in liposomes bearing positive charges ontheir surface (e.g., lipofectins) and (optionally) which are tagged withantibodies against cell surface antigens of the target tissue (Mizuno etal. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309;Japanese patent application 1047381; and European patent publicationEP-A-43075).

[0191] In clinical settings, the gene delivery systems for thetherapeutic MCH gene can be introduced into a patient by any of a numberof methods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by Stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057).

[0192] The pharmaceutical preparation of the gene therapy construct canconsist essentially of the gene delivery system in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery system can be produced in tact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

[0193] Antisense Therapy

[0194] Another aspect of the invention relates to the use of theisolated nucleic acid in “antisense” therapy. As used herein,“antisense” therapy refers to administration or in situ generation ofoligonucleotides or their derivatives which specifically hybridizes(e.g. binds) under cellular conditions, with the cellular mRNA and/orgenomic DNA encoding MCH so as to inhibit expression of the encodedprotein, e.g. by inhibiting transcription and/or translation. Thebinding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix. In general,“antisense” therapy refers to the range of techniques generally employedin the art, and includes any therapy which relies on specific binding tooligonucleotide sequences.

[0195] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thecell, produces RNA which is complementary to at least a unique portionof the cellular mRNA which encodes an MCH. Alternatively, the antisenseconstruct is an oligonucleotide probe which is generated ex vivo andwhich, when introduced into the cell causes inhibition of expression byhybridizing with the mRNA and/or genomic sequences of an MCH gene. Sucholigonucleotide probes are preferably modified oligonucleotide which areresistant to endogenous nucleases, e.g. exonucleases and/orendonucleases, and is therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by Van der Krol et al. (1988) Biotechniques6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

[0196] Accordingly, the modified oligomers of the invention are usefulin therapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of loads of administration,including systemic and topical or localized administration. For systemicadministration, injection is preferred, including intramuscular,intravenous, intraperitoneal, and subcutaneous for injection, theoligomers of the invention can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the oligomers may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included in the invention.

[0197] The compounds can be administered orally, or by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives, and detergents. Transmucosal administration may be throughnasal sprays or using suppositories. For oral administration, theoligomers are formulated into conventional oral administration formssuch as capsules, tablets, and tonics. For topical administration, theoligomers of the invention are formulated into ointments, salves, gels,or creams as known in the art.

[0198] In addition to use in therapy, the oligomers of the invention maybe used as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind.

[0199] Production of Fragments and Analogs

[0200] The inventor has discovered that MCH regulates eating behavior.Because structure of MCH is known, one skilled in the art can alter theMCH structure, e.g., by producing fragments or analogs, and test thenewly produced structures for activity. Examples of prior art methodswhich allow the production and testing of fragments and analogs arediscussed herein. These, or analogous methods can be used to make andscreen fragments and analogs of MCH that bind to naturally occurringligand of MCH, e.g., an MCH receptor, e.g., MC3-R. Likewise they can beused to make fragments and analogs that will bind MCH.

[0201] Generation of Fragments

[0202] Fragments of a protein can be produced in several ways, e.g.,recombinantly, by proteolytic digestion, or by chemical synthesis.Internal or terminal fragments of a polypeptide can be generated byremoving one or more nucleotides from one end (for a terminal fragment)or both ends (for an internal fragment) of a nucleic acid which encodesthe polypeptide. Expression of the mutagenized DNA produces polypeptidefragments. Digestion with “end-nibbling” endonucleases can thus generateDNA's which encode an array of fragments. DNA's which encode fragmentsof a protein can also be generated by random shearing, restrictiondigestion or a combination of the above-discussed methods.

[0203] Fragments can also be chemically synthesized using techniquesknown in the art such as conventional Merrifield solid phase f-Moc ort-Boc chemistry. For example, peptides of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

[0204] Production of Altered DNA and Peptide Sequences: Random Methods

[0205] Amino acid sequence variants of a protein can be prepared byrandom mutagenesis of DNA which encodes a protein or a particular domainor region of a protein. Useful methods include PCR mutagenesis andsaturation mutagenesis. A library of random amino acid sequence variantscan also be generated by the synthesis of a set of degenerateoligonucleotide sequences. (Methods for screening proteins in a libraryof variants are elsewhere herein.)

[0206] PCR Mutagenesis

[0207] In PCR mutagenesis, reduced Taq polymerase fidelity is used tointroduce random mutations into a cloned fragment of DNA (Leung et al.,1989, Technique 1:11-15). This is a very powerful and relatively rapidmethod of introducing random mutations. The DNA region to be mutagenizedis amplified using the polymerase chain reaction (PCR) under conditionsthat reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g.,by using a dGTP/dATP ratio of five and adding Mn²⁺ to the PCR reaction.The pool of amplified DNA fragments are inserted into appropriatecloning vectors to provide random mutant libraries.

[0208] Saturation Mutagenesis

[0209] Saturation mutagenesis allows for the rapid introduction of alarge number of single base substitutions into cloned DNA fragments(Mayers et al., 1985, Science 229:242). This technique includesgeneration of mutations, e.g., by chemical treatment or irradiation ofsingle-stranded DNA in vitro, and synthesis of a complimentary DNAstrand. The mutation frequency can be modulated by modulating theseverity of the treatment, and essentially all possible basesubstitutions can be obtained. Because this procedure does not involve agenetic selection for mutant fragments both neutral substitutions, aswell as those that alter function, are obtained. The distribution ofpoint mutations is not biased toward conserved sequence elements.

[0210] Degenerate Oligonucleotides

[0211] A library of homologs can also be generated from a set ofdegenerate oligonucleotide sequences. Chemical synthesis of a degeneratesequences can be carried out in an automatic DNA synthesizer, and thesynthetic genes then ligated into an appropriate expression vector. Thesynthesis of degenerate oligonucleotides is known in the art (see forexample, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981)Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AGWalton, Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477. Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

[0212] Production of Altered DNA and Peptide Sequences: Methods forDirected Mutagenesis

[0213] Non-random or directed, mutagenesis techniques can be used toprovide specific sequences or mutations in specific regions. Thesetechniques can be used to create variants which include, e.g.,deletions, insertions, or substitutions, of residues of the known aminoacid sequence of a protein. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconserved amino acids and then with more radical choices depending uponresults achieved, (2) deleting the target residue, or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

[0214] Alanine Scanning Mutagenesis

[0215] Alanine scanning mutagenesis is a useful method foridentification of certain residues or regions of the desired proteinthat are preferred locations or domains for mutagenesis, Cunningham andWells (Science 244:1081-1085, 1989). In alanine scanning, a residue orgroup of target residues are identified (e.g., charged residues such asArg, Asp, His, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine). Replacementof an amino acid can affect the interaction of the amino acids with thesurrounding aqueous environment in or outside the cell. Those domainsdemonstrating functional sensitivity to the substitutions are thenrefined by introducing further or other variants at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, alanine scanning or random mutagenesis may beconducted at the target codon or region and the expressed desiredprotein subunit variants are screened for the optimal combination ofdesired activity.

[0216] Oligonucleotide-Mediated Mutagenesis

[0217] Oligonucleotide-mediated mutagenesis is a useful method forpreparing substitution, deletion, and insertion variants of DNA, see,e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA isaltered by hybridizing an oligonucleotide encoding a mutation to a DNAtemplate, where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or native DNA sequence of thedesired protein. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will code for theselected alteration in the desired protein DNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Natl. Acad. Sci. USA, 75: 5765[1978]).

[0218] Cassette Mutagenesis

[0219] Another method for preparing variants, cassette mutagenesis, isbased on the technique described by Wells et al. (Gene, 34:315[1985]).The starting material is a plasmid (or other vector) which includes theprotein subunit DNA to be mutated. The codon(s) in the protein subunitDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the desired protein subunit DNA. Afterthe restriction sites have been introduced into the plasmid, the plasmidis cut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are comparable with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated desired protein subunit DNAsequence.

[0220] Combinatorial Mutagenesis

[0221] Combinatorial mutagenesis can also be used to generate mutants(Ladner et al., WO 88/06630). In this method, the amino acid sequencesfor a group of homologs or other related proteins are aligned,preferably to promote the highest homology possible. All of the aminoacids which appear at a given position of the aligned sequences can beselected to create a degenerate set of combinatorial sequences. Thevariegated library of variants is generated by combinatorial mutagenesisat the nucleic acid level, and is encoded by a variegated gene library.For example, a mixture of synthetic oligonucleotides can beenzymatically ligated into gene sequences such that the degenerate setof potential sequences are expressible as individual peptides, oralternatively, as a set of larger fusion proteins containing the set ofdegenerate sequences.

[0222] Primary High-Through-Put Methods for Screening Libraries ofPeptide Fragments or Homologs

[0223] Various techniques are known in the art for screening generatedmutant gene products. Techniques for screening large gene librariesoften include cloning the gene library into replicable expressionvectors, transforming appropriate cells with the resulting library ofvectors, and expressing the genes under conditions in which detection ofa desired activity, e.g., in this case, binding to MCH or naturallyoccuring ligand of MCH, e.g., an MCH receptor, e.g., MC3-R, facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Each of the techniques described below is amenable to highthrough-put analysis for screening large numbers of sequences created,e.g., by random mutagenesis techniques.

[0224] Two Hybrid Systems

[0225] Two hybrid assays (as with the other screening methods describedherein), can be used to identify fragments or analogs of a MCHpolypeptide which binds to naturally occuring ligand of MCH, e.g., anMCH receptor, e.g., MC3-R. These may include agonists, superagonists,and antagonists. (The MCH ligand is used as the bait protein and thelibrary of variants are expressed as fish fusion proteins.) In ananalogous fashion, a two hybrid assay (as with the other screeningmethods described herein), can be used to find fragments and analogswhich bind to MCH.

[0226] Display Libraries

[0227] In one approach to screening assays, the candidate peptides aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an appropriate receptorprotein via the displayed product is detected in a “panning assay”. Forexample, the gene library can be cloned into the gene for a surfacemembrane protein of a bacterial cell, and the resulting fusion proteindetected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991)Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).In a similar fashion, a detectably labeled ligand can be used to scorefor potentially functional peptide homologs. Fluorescently labeledligands, e.g., receptors, can be used to detect homolog which retainligand-binding activity. The use of fluorescently labeled ligands,allows cells to be visually inspected and separated under a fluorescencemicroscope, or, where the morphology of the cell permits, to beseparated by a fluorescence-activated cell sorter.

[0228] A gene library can be expressed as a fusion protein on thesurface of a viral particle. For instance, in the filamentous phagesystem, foreign peptide sequences can be expressed on the surface ofinfectious phage, thereby conferring two significant benefits. First,since these phage can be applied to affinity matrices at concentrationswell over 10¹³ phage per milliliter, a large number of phage can bescreened at one time. Second, since each infectious phage displays agene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd., and fl are most often used in phage display libraries.Either of the phage gIII or gVIII coat proteins can be used to generatefusion proteins without disrupting the ultimate packaging of the viralparticle. Foreign epitopes can be expressed at the NH₂-terminal end ofpIII and phage bearing such epitopes recovered from a large excess ofphage lacking this epitope (Ladner et al. PCT publication WO 90/02909;Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J.Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J. 12:725-734;Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

[0229] A common approach uses the maltose receptor of E. coli (the outermembrane protein, LamB) as a peptide fusion partner (Charbit et al.(1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted intoplasmids encoding the LamB gene to produce peptides fused into one ofthe extracellular loops of the protein. These peptides are available forbinding to ligands, e.g., to antibodies, and can elicit an immuneresponse when the cells are administered to animals. Other cell surfaceproteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392),PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al.(1991) Bio/Tech 9, 1369-1372), as well as large bacterial surfacestructures have served as vehicles for peptide display. Peptides can befused to pilin, a protein which polymerizes to form the pilus-a conduitfor interbacterial exchange of genetic information (Thiry et al. (1989)Appl. Environ. Microbiol 55, 984-993). Because of its role ininteracting with other cells, the pilus provides a useful support forthe presentation of peptides to the extracellular environment. Anotherlarge surface structure used for peptide display is the bacterial motiveorgan, the flagellum. Fusion of peptides to the subunit proteinflagellin offers a dense array of may peptides copies on the host cells(Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins ofother bacterial species have also served as peptide fusion partners.Examples include the Staphylococcus protein A and the outer membraneprotease IgA of Neisseria (Hansson et al. (1992) J. Bacteriol. 174,42394245 and Klauser et al. (1990) EMBO J. 9, 1991-1999).

[0230] In the filamentous phage systems and the LamB system describedabove, the physical link between the peptide and its encoding DNA occursby the containment of the DNA within a particle (cell or phage) thatcarries the peptide on its surface. Capturing the peptide captures theparticle and the DNA within. An alternative scheme uses the DNA-bindingprotein LacI to form a link between peptide and DNA (Cull et al. (1992)PNAS USA 89:1865-1869). This system uses a plasmid containing the LacIgene with an oligonucleotide cloning site at its 3′-end. Under thecontrolled induction by arabinose, a LacI-peptide fusion protein isproduced. This fusion retains the natural ability of LacI to bind to ashort DNA sequence known as LacO operator (LacO). By installing twocopies of LacO on the expression plasmid, the LacI-peptide fusion bindstightly to the plasmid that encoded it. Because the plasmids in eachcell contain only a single oligonucleotide sequence and each cellexpresses only a single peptide sequence, the peptides becomespecifically and stably associated with the DNA sequence that directedits synthesis. The cells of the library are gently lysed and thepeptide-DNA complexes are exposed to a matrix of immobilized receptor torecover the complexes containing active peptides. The associated plasmidDNA is then reintroduced into cells for amplification and DNA sequencingto determine the identity of the peptide ligands. As a demonstration ofthe practical utility of the method, a large random library ofdodecapeptides was made and selected on a monoclonal antibody raisedagainst the opioid peptide dynorphin B. A cohort of peptides wasrecovered, all related by a consensus sequence corresponding to asix-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl.Acad. Sci. U.S.A. 89-1869)

[0231] This scheme, sometimes referred to as peptides-on-plasmids,differs in two important ways from the phage display methods. First, thepeptides are attached to the C-terminus of the fusion protein, resultingin the display of the library members as peptides having free carboxytermini. Both of the filamentous phage coat proteins, pIII and pVIII,are anchored to the phage through their C-termini, and the guestpeptides are placed into the outward-extending N-terminal domains. Insome designs, the phage-displayed peptides are presented right at theamino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl.Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set ofbiological biases affecting the population of peptides actually presentin the libraries. The LacI fusion molecules are confined to thecytoplasm of the host cells. The phage coat fusions are exposed brieflyto the cytoplasm during translation but are rapidly secreted through theinner membrane into the periplasmic compartment, remaining anchored inthe membrane by their C-terminal hydrophobic domains, with theN-termini, containing the peptides, protruding into the periplasm whileawaiting assembly into phage particles. The peptides in the LacI andphage libraries may differ significantly as a result of their exposureto different proteolytic activities. The phage coat proteins requiretransport across the inner membrane and signal peptidase processing as aprelude to incorporation into phage. Certain peptides exert adeleterious effect on these processes and are underrepresented in thelibraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). Theseparticular biases are not a factor in the LacI display system.

[0232] The number of small peptides available in recombinant randomlibraries is enormous. Libraries of 10⁷-10⁹ independent clones areroutinely prepared. Libraries as large as 10¹¹ recombinants have beencreated, but this size approaches the practical limit for clonelibraries. This limitation in library size occurs at the step oftransforming the DNA containing randomized segments into the hostbacterial cells. To circumvent this limitation, an in vitro system basedon the display of nascent peptides in polysome complexes has recentlybeen developed. This display library method has the potential ofproducing libraries 3-6 orders of magnitude larger than the currentlyavailable phage/phagemid or plasmid libraries. Furthermore, theconstruction of the libraries, expression of the peptides, andscreening, is done in an entirely cell-free format.

[0233] In one application of this method (Gallop et al. (1994) J. Med.Chem. 37(9):1233-1251), a molecular DNA library encoding 10¹²decapeptides was constructed and the library expressed in an E. coli S30in vitro coupled transcription/translation system. Conditions werechosen to stall the ribosomes on the mRNA, causing the accumulation of asubstantial proportion of the RNA in polysomes and yielding complexescontaining nascent peptides still linked to their encoding RNA. Thepolysomes are sufficiently robust to be affinity purified on immobilizedreceptors in much the same way as the more conventional recombinantpeptide display libraries are screened. RNA from the bound complexes isrecovered, converted to cDNA, and amplified by PCR to produce a templatefor the next round of synthesis and screening. The polysome displaymethod can be coupled to the phage display system. Following severalrounds of screening, cDNA from the enriched pool of polysomes was clonedinto a phagemid vector. This vector serves as both a peptide expressionvector, displaying peptides fused to the coat proteins, and as a DNAsequencing vector for peptide identification. By expressing thepolysome-derived peptides on phage, one can either continue the affinityselection procedure in this format or assay the peptides on individualclones for binding activity in a phage ELISA, or for binding specificityin a completion phage ELISA (Barret, et al. (1992) Anal. Biochem204,357-364). To identify the sequences of the active peptides onesequences the DNA produced by the phagemid host.

[0234] Secondary Screens

[0235] The high through-put assays described above can be followed bysecondary screens in order to identify further biological activitieswhich will, e.g., allow one skilled in the art to differentiate agonistsfrom antagonists. The type of a secondary screen used will depend on thedesired activity that needs to be tested (some of the assays which testspecific MCH activity have been described above). For example, an assaycan be developed in which the ability to inhibit an interaction betweena protein of interest and its respective ligand can be used to identifyantagonists from a group of peptide fragments isolated though one of theprimary screens described above.

[0236] Therefore, methods for generating fragments and analogs andtesting them for activity are known in the art. Once the core sequenceof interest is identified, it is routine to perform for one skilled inthe art to obtain analogs and fragments.

[0237] Peptide Mimetics

[0238] The invention also provides for reduction of the protein bindingdomains of the MCH peptide, to generate mimetics, e.g. peptide ornon-peptide agents, which are able to disrupt binding, in this case, ofan MCH with a naturally occurring ligand of MCH, e.g., an MCH receptor,e.g., MC3-R. The critical residues of the MCH peptide which are involvedin molecular recognition of MCH ligand can be determined and used togenerate MCH-derived peptidomimetics which competitively ornon-competatively inhibit binding of the MCH with an MCH ligand (see,for example, “Peptide inhibitors of human papillomavirus protein bindingto retinoblastoma gene protein” European patent applications EP-412,762Aand EP-B31,080A). By employing, for example, scanning mutagenesis to mapthe amino acid residues of a particular MCH peptide involved in bindingthe MCH ligand, peptidomimetic compounds (e.g. diazepine or isoquinolinederivatives) can be generated which mimic those residues in binding toan MCH ligand, and which therefore can inhibit binding of the MCH to theligand and thereby interfere with the function of MCH. For instance,non-hydrolyzable peptide analogs of such residues can be generated usingbenzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), azepine (e.g., see Huffinan et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), substituted gama lactam rings (Garvey et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986)J Med Chem 29:295; and Ewenson et al. in Peptides: Structure andFunction (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc PerkinTrans 1:1231), and β-aminoalcohols (Gordon et al. (1985) Biochem BiophysRes Commun 126:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71).

[0239] Administration

[0240] The compounds of the invention can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention cross the BBB, they can beformulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs (“targetingmoieties”), thus providing targeted drug delivery (see, e.g., V. V.Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moietiesinclude folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low etal.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);gp120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273. In a preferred embodiment, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety.

[0241] To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a subjectin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., (1984) J. Neuroimmunol.7:27).

[0242] The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

[0243] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fingi. The vehicle can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

[0244] Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0245] Appropriate dosages can be determined by prior art methods, butcan be in the range of 0.001-100 mg/kg, or 0.1-10 mg/kg of body weight.

Other Embodiments

[0246] Analogs can differ from naturally occurring MCH in amino acidsequence or in ways that do not involve sequence, or both. Non-sequencemodifications include in vivo or in vitro chemical derivatization ofMCH. Non-sequence modifications include changes in acetylation,methylation, phosphorylation, carboxylation, or glycosylation.

[0247] Preferred analogs include MCH (or biologically active fragmentsthereof) whose sequences differ from the wild-type sequence by one ormore conservative amino acid substitutions or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not abolish the MCH biological activity. Conservativesubstitutions typically include the substitution of one amino acid foranother with similar characteristics, e.g., substitutions within thefollowing groups: valine, glycine; glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Otherconservative substitutions can be taken from the table below. TABLE 1CONSERVATIVE AMINO ACID REPLACEMENTS For Amino Acid Code Replace withany of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg,Lys, D-Lys, homo-Arg, D- homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-OrnAsparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid DD-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys,Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp,D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln GlycineG Ala, D-Ala, Pro, D-Pro, β-Ala Acp Isoleucine I D-Ile, Val, D-Val, Leu,D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-MetLysine K D-Lys, Arg, D-Arg, homo-Arg, D- homo-Arg, Met, D-Met, Ile,D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu,Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp,D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylprolineProline P D-Pro, L-I-thioazolidine-4- carboxylic acid, D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met,D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser,allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr,Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile,Met, D-Met

[0248] Other analogs within the invention are those with modificationswhich increase peptide stability; such analogs may contain, for example,one or more non-peptide bonds (which replace the peptide bonds) in thepeptide sequence. Also included are: analogs that include residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., β or γ aminoacids; and cyclic analogs.

[0249] In order to obtain an MCH polypeptide, MCH-encoding DNA can beintroduced into an expression vector, the vector introduced into a cellsuitable for expression of the desired protein, and the peptiderecovered and purified, by prior art methods. Antibodies to the peptidesan proteins can be made by immunizing an animal, e.g., a rabbit ormouse, and recovering anti-MCH antibodies by prior art methods.

[0250] Other embodiments are within the following claims.

What is claimed is: 1-29 (Canceled).
 30. A method of inhibitingappetite, or the gain of weight, in a subject comprising: identifying asubject in need of inhibiting appetite or weight gain; and administeringan effective amount of an antagonist of melanocyte concentrating hormone(MCH) to said subject, wherein the antagonist binds an MCH receptor. 31.The method of claim 30, wherein the antagonist binds competitively to anMCH receptor.
 32. The method of claim 30, wherein the antagonist bindsnon-competitively to an MCH receptor.
 33. The method of claim 30,wherein the antagonist is a peptide analog of MCH.
 34. The method ofclaim 33, wherein the peptide analog has 1, 2, 3, 4, 5 or more residueswithin the MCH ring structure modified or substituted with anonconserved amino acid.
 35. The method of claim 34, wherein between 1and 5 residues within the MCH ring structure modified or substitutedwith a nonconserved amino acid.
 36. The method of claim 33, wherein thepeptide analog is deleted for any or all of the residues between R1 andR6 or between R18 and R19.
 37. The method of claim 34, wherein thepeptide analog is deleted for any or all of the residues between R1 andR6 or between R18 and R19.
 38. The method of claim 35, wherein thepeptide analog is deleted for any or all of the residues between R1 andR6 or between R18 and R19.
 39. The method of claim 33, wherein thepeptide analog has least 50% homology with MCH.
 40. The method of claim33, wherein the peptide analog has least 60% homology with MCH.
 41. Themethod of claim 33, wherein the peptide analog has least 70% homologywith MCH.
 42. The method of claim 33, wherein the peptide analog hasleast 80% homology with MCH.
 43. The method of claim 33, wherein thepeptide analog has least 90% homology with MCH.